Malic enzyme in the rat liver is under the control of both hormonal and nutritional stimuli. Thyroid hormone levels are known to dramatically affect the intracellular concentration of malic enzyme. Feeding an animal with a diet high in carbohydrate and low in fat, conditions which favor lipogenesis, also leads to an induction of malic enzyme. The experiments proposed are designed to examine the mechanisms responsible for these alterations. Primary emphasis will be placed on measurements of the messenger RNA coding for malic enzyme. For this purpose, two methods will be used: an in vitro translational assay and a molecular hybridization assay. For the latter assay, it will be necessary to isolate complementary DNA sequences to the malic enzyme mRNA. This isolation will be achieved by molecular cloning of a partially purified preparation of cDNA to malic enzyme, followed by screening of bacterial transformants for the presence of appropriate DNA sequences. Levels of messenger RNA for malic enzyme will be monitored in livers of animals after induction with either thyroid hormone or high carbohydrate, fat free diet. Dose-response relationships and time courses will be established in an effort to characterize the responses mechanistically. Additional studies will be performed in isolated hepatocytes in which various hormonal and dietary factors can be manipulated under defined conditions. Attempts will be made to define the sites of regulation of mRNA production. The synthesis of malic enzyme mRNA sequences in isolated nuclei will be measured. These studies will lead to a better understanding of the regulation of malic enzyme synthesis, especially at the level of mRNA production. Important information on the mechanisms of action of thyroid hormones and of dietary factors will hopefully be elucidated.